bst dna polymerase  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Bst DNA Polymerase Full Length
    Description:
    Bst DNA Polymerase Full Length 500 units
    Catalog Number:
    m0328s
    Price:
    70
    Size:
    500 units
    Category:
    Thermostable DNA Polymerases
    Buy from Supplier


    Structured Review

    New England Biolabs bst dna polymerase
    Bst DNA Polymerase Full Length
    Bst DNA Polymerase Full Length 500 units
    https://www.bioz.com/result/bst dna polymerase/product/New England Biolabs
    Average 99 stars, based on 102 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    2) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability"

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    3) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability "

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    4) Product Images from "Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method"

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08067-x

    Hinge-initiated primer-based replication with one hinge-primer and PCR-like primer. The hinge-primer anneals to the target region and will be extended to double strand DNA by Bst DNA polymerase (1). The refolding to the thermodynamically more stable hairpin structure liberates the initial priming site (2–3). These steps are repeated and the sense DNA strand will be released. The PCR-like primer can anneal to the priming site and will be extended (4).
    Figure Legend Snippet: Hinge-initiated primer-based replication with one hinge-primer and PCR-like primer. The hinge-primer anneals to the target region and will be extended to double strand DNA by Bst DNA polymerase (1). The refolding to the thermodynamically more stable hairpin structure liberates the initial priming site (2–3). These steps are repeated and the sense DNA strand will be released. The PCR-like primer can anneal to the priming site and will be extended (4).

    Techniques Used: Polymerase Chain Reaction

    Mechanism of the symmetric Hinge-initiated primer-dependent replication with two hinge-primer. The first stage (A) starts with initial annealing ( 1 ) of the forward hinge-primer, followed by extension to double strand DNA by Bst DNA polymerase ( 2 ) and refolding to the thermodynamically more stable hairpin structure which liberates ( 3 ) the initial priming site. These steps are repeated following the release of the sense DNA strand ( 4 ). The second stage (B) starts with annealing of the reverse hinge-primer to the newly generated and released single strand ( 5 ) followed by the extension and refolding to a second hairpin structure at both ends ( 6 ). The initial priming sites on the sense and anti-sense strands are thus liberated by refolding ( 7 ). Due to continuous recycling of hinge-primer binding sites, the reaction finally results in DNA product accumulation for specific detection. The “X” in the primer represents a blocking modification for the Bst DNA polymerase. Examples are a dSpacer (abasic furan) or an C12-Spacer (hexaethylenglycol). Optionally, an additional outer primer can be applied to increase the speed of initial single strand template generation.
    Figure Legend Snippet: Mechanism of the symmetric Hinge-initiated primer-dependent replication with two hinge-primer. The first stage (A) starts with initial annealing ( 1 ) of the forward hinge-primer, followed by extension to double strand DNA by Bst DNA polymerase ( 2 ) and refolding to the thermodynamically more stable hairpin structure which liberates ( 3 ) the initial priming site. These steps are repeated following the release of the sense DNA strand ( 4 ). The second stage (B) starts with annealing of the reverse hinge-primer to the newly generated and released single strand ( 5 ) followed by the extension and refolding to a second hairpin structure at both ends ( 6 ). The initial priming sites on the sense and anti-sense strands are thus liberated by refolding ( 7 ). Due to continuous recycling of hinge-primer binding sites, the reaction finally results in DNA product accumulation for specific detection. The “X” in the primer represents a blocking modification for the Bst DNA polymerase. Examples are a dSpacer (abasic furan) or an C12-Spacer (hexaethylenglycol). Optionally, an additional outer primer can be applied to increase the speed of initial single strand template generation.

    Techniques Used: Generated, Binding Assay, Blocking Assay, Modification

    5) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    6) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    7) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    8) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    9) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    10) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    11) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    12) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    13) Product Images from "A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions"

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp037

    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.
    Figure Legend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Techniques Used: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

    14) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    15) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    16) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    17) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    18) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    19) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    20) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    21) Product Images from "High-efficiency and integrable DNA arithmetic and logic system based on strand displacement synthesis"

    Article Title: High-efficiency and integrable DNA arithmetic and logic system based on strand displacement synthesis

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13310-2

    Construction of six basic dual-rail systems and their performance. a Construction of the AND, OR, NAND, NOR, XOR, and XNOR dual-rail gates. The upper regular octagon shows a single-rail AND gate, and the lower regular hexagon shows a single-rail OR gate. The green and magenta colors mean defined TRUE and FALSE signals, respectively. Superscripts 1 and 0 also indicate the TRUE and FALSE inputs and outputs, respectively. When computations were performed, a set of input strands must be adder, e.g., inputs (1, 0) means adding inputs I A 1 (TURE) and I B 0 (FALSE). Detailed structures can be seen in Fig. S12. Reaction details of AND and XOR gates can be seen in Supplementary Figs. 7 and 8 . b , c Reaction kinetics of the dual-rail AND and XOR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment), TRI and FRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The FAM and ROX signals correspond to the TRUE and FALSE returns, respectively. Sequences of the DNA strands are listed in the Supplementary Information . d Summary of all the outputs computed by the six basic logic gates constructed from DNA.
    Figure Legend Snippet: Construction of six basic dual-rail systems and their performance. a Construction of the AND, OR, NAND, NOR, XOR, and XNOR dual-rail gates. The upper regular octagon shows a single-rail AND gate, and the lower regular hexagon shows a single-rail OR gate. The green and magenta colors mean defined TRUE and FALSE signals, respectively. Superscripts 1 and 0 also indicate the TRUE and FALSE inputs and outputs, respectively. When computations were performed, a set of input strands must be adder, e.g., inputs (1, 0) means adding inputs I A 1 (TURE) and I B 0 (FALSE). Detailed structures can be seen in Fig. S12. Reaction details of AND and XOR gates can be seen in Supplementary Figs. 7 and 8 . b , c Reaction kinetics of the dual-rail AND and XOR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment), TRI and FRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The FAM and ROX signals correspond to the TRUE and FALSE returns, respectively. Sequences of the DNA strands are listed in the Supplementary Information . d Summary of all the outputs computed by the six basic logic gates constructed from DNA.

    Techniques Used: Transferring, Construct

    Construction of DNA ALU with our DNA logic gates. a Abstract diagram of a typical ALU. b A typical construction of a 1-bit ALU with a digital logic circuit. A and B: inputs; C in : carry in; S 0 and S 1 : select signals; Y: output; C out : carry out. c Assembling the 4:1 multiplexer with DNA components. The sequences of the strands are listed in the Supplementary Table 2 . The details of the integrated logic gates are shown in Fig. S20. d Function table of the DNA ALU. e Summary of the outputs of the ALU. f , g Reaction kinetics of the ALU with all possible combinations of inputs. The reaction was performed with 12 U Bst polymerase (large fragment), TRIII, FRIII, TRII, and FRII. The curve was plotted by transferring the cycle values into the reaction time. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to the TRUE and FALSE returns of Y, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Fig. 22 . The sequences of the DNA strands are listed in the Supplementary Table 2 .
    Figure Legend Snippet: Construction of DNA ALU with our DNA logic gates. a Abstract diagram of a typical ALU. b A typical construction of a 1-bit ALU with a digital logic circuit. A and B: inputs; C in : carry in; S 0 and S 1 : select signals; Y: output; C out : carry out. c Assembling the 4:1 multiplexer with DNA components. The sequences of the strands are listed in the Supplementary Table 2 . The details of the integrated logic gates are shown in Fig. S20. d Function table of the DNA ALU. e Summary of the outputs of the ALU. f , g Reaction kinetics of the ALU with all possible combinations of inputs. The reaction was performed with 12 U Bst polymerase (large fragment), TRIII, FRIII, TRII, and FRII. The curve was plotted by transferring the cycle values into the reaction time. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to the TRUE and FALSE returns of Y, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Fig. 22 . The sequences of the DNA strands are listed in the Supplementary Table 2 .

    Techniques Used: Transferring

    Construction of the full adder and 4:1 multiplexer from our DNA logic gates. a , e A typical construction of the full adder and the 4:1 multiplexer with the digital logic circuit. D 0 –D 3 : inputs; S 0 and S 1 : select signals; Z: output. b , f Constructing a full adder and the 4:1 multiplexer with DNA logic gates. The sequences of the DNA strands are listed in the Supplementary table 2 . c , g Summary of all the outputs computed by the DNA full adder and 4:1 multiplexer. d , h Reaction kinetics of the full adder and the 4:1 multiplexer with all possible combinations of inputs. For the full adder, the reaction was performed with 12 U Bst polymerase (large fragment), TRI, FRI, TRII, and FRII. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. For the full adder: The TRI (FAM) and FRI (ROX) signals correspond to TRUE and FALSE returns of S, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. For the 4:1 multiplexer, the reaction was performed with 6.4 U Bst polymerase (large fragment), TRIII and FRII. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to TRUE and FALSE returns, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Figs. 14 and 19 .
    Figure Legend Snippet: Construction of the full adder and 4:1 multiplexer from our DNA logic gates. a , e A typical construction of the full adder and the 4:1 multiplexer with the digital logic circuit. D 0 –D 3 : inputs; S 0 and S 1 : select signals; Z: output. b , f Constructing a full adder and the 4:1 multiplexer with DNA logic gates. The sequences of the DNA strands are listed in the Supplementary table 2 . c , g Summary of all the outputs computed by the DNA full adder and 4:1 multiplexer. d , h Reaction kinetics of the full adder and the 4:1 multiplexer with all possible combinations of inputs. For the full adder, the reaction was performed with 12 U Bst polymerase (large fragment), TRI, FRI, TRII, and FRII. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. For the full adder: The TRI (FAM) and FRI (ROX) signals correspond to TRUE and FALSE returns of S, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. For the 4:1 multiplexer, the reaction was performed with 6.4 U Bst polymerase (large fragment), TRIII and FRII. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to TRUE and FALSE returns, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Figs. 14 and 19 .

    Techniques Used:

    Construction of AND and OR gates with DNA polymerase and their performance. a , c Right: structure of the AND and OR gates. The capital names are the sequence names; the lowercase names refer to the elementary sequences and asterisk indicates a complementary sequence. Each elementary sequence contains 18 bases. The gray parts are 4-mer spacer sequences used to reduce steric hinderance. The sequences of inputs A and B are the same as a and b. Left: abstract diagram of the AND and OR gates. The regular octagon shows the main structure of the DNA components; the two bold lines on the left indicate the binding sites of the two inputs in the DNA component; the vector line on the right indicates the potential output. b , d Mechanism of the AND and OR gates. e The fluorescent reporter used to visualize the devices. The letter Q denotes the quencher, and F denotes the fluorophore. f , g Reaction kinetics of the AND and OR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment) and TRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the relative fluorescence unit (RFU) values in the FAM channel with the highest signals. The original signals are plotted in Supplementary Fig. 3 . The sequences of the DNA strands are listed in the Supplementary Table 2 .
    Figure Legend Snippet: Construction of AND and OR gates with DNA polymerase and their performance. a , c Right: structure of the AND and OR gates. The capital names are the sequence names; the lowercase names refer to the elementary sequences and asterisk indicates a complementary sequence. Each elementary sequence contains 18 bases. The gray parts are 4-mer spacer sequences used to reduce steric hinderance. The sequences of inputs A and B are the same as a and b. Left: abstract diagram of the AND and OR gates. The regular octagon shows the main structure of the DNA components; the two bold lines on the left indicate the binding sites of the two inputs in the DNA component; the vector line on the right indicates the potential output. b , d Mechanism of the AND and OR gates. e The fluorescent reporter used to visualize the devices. The letter Q denotes the quencher, and F denotes the fluorophore. f , g Reaction kinetics of the AND and OR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment) and TRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the relative fluorescence unit (RFU) values in the FAM channel with the highest signals. The original signals are plotted in Supplementary Fig. 3 . The sequences of the DNA strands are listed in the Supplementary Table 2 .

    Techniques Used: Sequencing, Binding Assay, Plasmid Preparation, Transferring, Fluorescence

    22) Product Images from "Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification"

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001948

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Figure Legend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Techniques Used: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.
    Figure Legend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Techniques Used: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.
    Figure Legend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Techniques Used: Lamp Assay, Infection, Isolation

    23) Product Images from "Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues"

    Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-7-312

    Detection of Skp2 amplification in NSCLC samples following whole genome amplification by Bst DNA polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.
    Figure Legend Snippet: Detection of Skp2 amplification in NSCLC samples following whole genome amplification by Bst DNA polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.

    Techniques Used: Amplification, Whole Genome Amplification

    Gel electrophoresis of Bst DNA polymerase amplification products . From left to right: (1) Lambda DNA-Hind III digested ladder; FFPE samples: (2, 3) normal lung 3 4; (4, 5) neuroblastoma xenografts LAN-5 SK-N-BE (2) and (6, 7) NSCLC 3 4; (8) Commercial DNA; (9) Negative control. Samples were analyzed in 0.5% agarose gel, stained with SYBR-green II. 10% by volume of the amplification product was used for the gel electrophoresis.
    Figure Legend Snippet: Gel electrophoresis of Bst DNA polymerase amplification products . From left to right: (1) Lambda DNA-Hind III digested ladder; FFPE samples: (2, 3) normal lung 3 4; (4, 5) neuroblastoma xenografts LAN-5 SK-N-BE (2) and (6, 7) NSCLC 3 4; (8) Commercial DNA; (9) Negative control. Samples were analyzed in 0.5% agarose gel, stained with SYBR-green II. 10% by volume of the amplification product was used for the gel electrophoresis.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Lambda DNA Preparation, Formalin-fixed Paraffin-Embedded, Negative Control, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    N- myc gene content in Bst amplified vs. non-amplified neuroblastoma xenografts . For neuroblastoma xenografts, where N- myc gene is highly amplified, relative gene content in Bst amplified samples was comparable to the respective values in non-amplified samples and the representational distortion was negligible. Note: NBL-S is a neuroblastoma cell line that lacks N- myc amplification and appropriately the calculated copy numbers were 1.12 ± 0.03 for non-amplified DNA and 1.14 ± 0.35 for Bst amplified DNA. Error bars represent SD.
    Figure Legend Snippet: N- myc gene content in Bst amplified vs. non-amplified neuroblastoma xenografts . For neuroblastoma xenografts, where N- myc gene is highly amplified, relative gene content in Bst amplified samples was comparable to the respective values in non-amplified samples and the representational distortion was negligible. Note: NBL-S is a neuroblastoma cell line that lacks N- myc amplification and appropriately the calculated copy numbers were 1.12 ± 0.03 for non-amplified DNA and 1.14 ± 0.35 for Bst amplified DNA. Error bars represent SD.

    Techniques Used: Amplification

    Mean amplification of DNA by Bst polymerase . All reactions started with 10 ng of target DNA. FFPE samples: Lung 1–5, neuroblastoma xenografts (LAN-5, NUB-7, SK-N-BE(2), NBL-S) and NSCLC 1–7. Intact DNA samples: FL (Frozen Lung) 1–4 and Positive C. (Control). Negative C. (Control) contained water in lieu of target DNA. For each sample the mean and SD of 2–6 independent experiments is shown.
    Figure Legend Snippet: Mean amplification of DNA by Bst polymerase . All reactions started with 10 ng of target DNA. FFPE samples: Lung 1–5, neuroblastoma xenografts (LAN-5, NUB-7, SK-N-BE(2), NBL-S) and NSCLC 1–7. Intact DNA samples: FL (Frozen Lung) 1–4 and Positive C. (Control). Negative C. (Control) contained water in lieu of target DNA. For each sample the mean and SD of 2–6 independent experiments is shown.

    Techniques Used: Amplification, Formalin-fixed Paraffin-Embedded

    24) Product Images from "Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii"

    Article Title: Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2017.e00415

    Lyophilized LAMP reagents for the detection of Coxiella burnetii . The lyophilized reagents were inside a 0.2 ml vial containing Bst DNA polymerase, SYBR green, primers, and dNTPs. Vials were packaged in a zipped aluminum foil bag.
    Figure Legend Snippet: Lyophilized LAMP reagents for the detection of Coxiella burnetii . The lyophilized reagents were inside a 0.2 ml vial containing Bst DNA polymerase, SYBR green, primers, and dNTPs. Vials were packaged in a zipped aluminum foil bag.

    Techniques Used: SYBR Green Assay

    25) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    26) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    Related Articles

    Produced:

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ). ..

    Amplification:

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability
    Article Snippet: .. 2.2 Viral RNA amplified by RT-LAMP Five microliters of extracted RNA (the same amount for regular RT-PCR) was taken for RT-LAMP amplification in a 25-μL mixture consisting of Bst DNA polymerase (New England Biolab) 8 U, 10× thermal buffer 1.0 μL, AMV reverse transcriptase (Fermentas) 5 U, 5× AMV first-strand buffer 4.0 μL, 25 μM betaine (Sigma, USA) 1.0 μL, 2.5 mmol/L dNTP mixtures 5 μL, 0.1% Triton X-100 0.25 μL, and a primer mixture, including 0.2 μM F3 and B3, and 1.6 μM FIP and BIP. .. RT-LAMP primer sets were designed using the Primer Explorer V3.0 online software ( http://primerexplorer.jp/v3_manual/index.html ) and manually selected according to the principle for RT-LAMP ( and ).

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. In this work, we showed that as the concentration of Bst DNA polymerase was increased from 4 U to 8 U, the amplification improved dramatically, however, the tendency of amplification efficiency improvement from 6 U to 8 U is far less than that of 4 U to 6 U (Figs and ). .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ).

    Purification:

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
    Article Snippet: .. LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template. .. Hinge-assay Hinge-amplification assays were set up in a total volume of 12.5 µL containing 1x Thermo Pol® Reaction buffer, 8 mM magnesium chloride, 0.8 M betaine, 1.0 mM of dNTP-mix, 0.32 U/µL Bst 3.0 DNA Polymerase, 0.8x SYBR Green I.

    SYBR Green Assay:

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
    Article Snippet: .. LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template. .. Hinge-assay Hinge-amplification assays were set up in a total volume of 12.5 µL containing 1x Thermo Pol® Reaction buffer, 8 mM magnesium chloride, 0.8 M betaine, 1.0 mM of dNTP-mix, 0.32 U/µL Bst 3.0 DNA Polymerase, 0.8x SYBR Green I.

    Concentration Assay:

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ). ..

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. In this work, we showed that as the concentration of Bst DNA polymerase was increased from 4 U to 8 U, the amplification improved dramatically, however, the tendency of amplification efficiency improvement from 6 U to 8 U is far less than that of 4 U to 6 U (Figs and ). .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ).

    Incubation:

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: .. NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs). .. After further incubation at 37°C for 20 min, DNA was purified by phenol/chloroform extraction and ethanol precipitation, and subjected to cccDNA-selective qPCR or RCA, as described above.

    other:

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability
    Article Snippet: Without Bst DNA polymerase or RNA templates, RT-LAMP was unable to amplify RNA ( A).

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: The quantity of Bst DNA polymerase in the reaction mixture was set at 2.0 U, 4.0 U, 6.0 U and 8.0 U. Mg2+ concentrations were set to 5.0 mM, 6.0 mM, 7.0 mM and 8.0 mM, respectively (Fig. ).

    Lamp Assay:

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
    Article Snippet: .. LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template. .. Hinge-assay Hinge-amplification assays were set up in a total volume of 12.5 µL containing 1x Thermo Pol® Reaction buffer, 8 mM magnesium chloride, 0.8 M betaine, 1.0 mM of dNTP-mix, 0.32 U/µL Bst 3.0 DNA Polymerase, 0.8x SYBR Green I.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability
    Article Snippet: .. 2.2 Viral RNA amplified by RT-LAMP Five microliters of extracted RNA (the same amount for regular RT-PCR) was taken for RT-LAMP amplification in a 25-μL mixture consisting of Bst DNA polymerase (New England Biolab) 8 U, 10× thermal buffer 1.0 μL, AMV reverse transcriptase (Fermentas) 5 U, 5× AMV first-strand buffer 4.0 μL, 25 μM betaine (Sigma, USA) 1.0 μL, 2.5 mmol/L dNTP mixtures 5 μL, 0.1% Triton X-100 0.25 μL, and a primer mixture, including 0.2 μM F3 and B3, and 1.6 μM FIP and BIP. .. RT-LAMP primer sets were designed using the Primer Explorer V3.0 online software ( http://primerexplorer.jp/v3_manual/index.html ) and manually selected according to the principle for RT-LAMP ( and ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs bst dna polymerase
    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the <t>Bst</t> <t>DNA</t> polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Bst Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase/product/New England Biolabs
    Average 99 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Journal: Scientific Reports

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    doi: 10.1038/s41598-017-13881-4

    Figure Lengend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Article Snippet: In this work, we showed that as the concentration of Bst DNA polymerase was increased from 4 U to 8 U, the amplification improved dramatically, however, the tendency of amplification efficiency improvement from 6 U to 8 U is far less than that of 4 U to 6 U (Figs and ).

    Techniques: Amplification, Concentration Assay

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Journal: Diagnostic Microbiology and Infectious Disease

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    doi: 10.1016/j.diagmicrobio.2011.07.014

    Figure Lengend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Article Snippet: 2.2 Viral RNA amplified by RT-LAMP Five microliters of extracted RNA (the same amount for regular RT-PCR) was taken for RT-LAMP amplification in a 25-μL mixture consisting of Bst DNA polymerase (New England Biolab) 8 U, 10× thermal buffer 1.0 μL, AMV reverse transcriptase (Fermentas) 5 U, 5× AMV first-strand buffer 4.0 μL, 25 μM betaine (Sigma, USA) 1.0 μL, 2.5 mmol/L dNTP mixtures 5 μL, 0.1% Triton X-100 0.25 μL, and a primer mixture, including 0.2 μM F3 and B3, and 1.6 μM FIP and BIP.

    Techniques: Amplification, Marker, Electrophoresis

    Hinge-initiated primer-based replication with one hinge-primer and PCR-like primer. The hinge-primer anneals to the target region and will be extended to double strand DNA by Bst DNA polymerase (1). The refolding to the thermodynamically more stable hairpin structure liberates the initial priming site (2–3). These steps are repeated and the sense DNA strand will be released. The PCR-like primer can anneal to the priming site and will be extended (4).

    Journal: Scientific Reports

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

    doi: 10.1038/s41598-017-08067-x

    Figure Lengend Snippet: Hinge-initiated primer-based replication with one hinge-primer and PCR-like primer. The hinge-primer anneals to the target region and will be extended to double strand DNA by Bst DNA polymerase (1). The refolding to the thermodynamically more stable hairpin structure liberates the initial priming site (2–3). These steps are repeated and the sense DNA strand will be released. The PCR-like primer can anneal to the priming site and will be extended (4).

    Article Snippet: LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template.

    Techniques: Polymerase Chain Reaction

    Mechanism of the symmetric Hinge-initiated primer-dependent replication with two hinge-primer. The first stage (A) starts with initial annealing ( 1 ) of the forward hinge-primer, followed by extension to double strand DNA by Bst DNA polymerase ( 2 ) and refolding to the thermodynamically more stable hairpin structure which liberates ( 3 ) the initial priming site. These steps are repeated following the release of the sense DNA strand ( 4 ). The second stage (B) starts with annealing of the reverse hinge-primer to the newly generated and released single strand ( 5 ) followed by the extension and refolding to a second hairpin structure at both ends ( 6 ). The initial priming sites on the sense and anti-sense strands are thus liberated by refolding ( 7 ). Due to continuous recycling of hinge-primer binding sites, the reaction finally results in DNA product accumulation for specific detection. The “X” in the primer represents a blocking modification for the Bst DNA polymerase. Examples are a dSpacer (abasic furan) or an C12-Spacer (hexaethylenglycol). Optionally, an additional outer primer can be applied to increase the speed of initial single strand template generation.

    Journal: Scientific Reports

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

    doi: 10.1038/s41598-017-08067-x

    Figure Lengend Snippet: Mechanism of the symmetric Hinge-initiated primer-dependent replication with two hinge-primer. The first stage (A) starts with initial annealing ( 1 ) of the forward hinge-primer, followed by extension to double strand DNA by Bst DNA polymerase ( 2 ) and refolding to the thermodynamically more stable hairpin structure which liberates ( 3 ) the initial priming site. These steps are repeated following the release of the sense DNA strand ( 4 ). The second stage (B) starts with annealing of the reverse hinge-primer to the newly generated and released single strand ( 5 ) followed by the extension and refolding to a second hairpin structure at both ends ( 6 ). The initial priming sites on the sense and anti-sense strands are thus liberated by refolding ( 7 ). Due to continuous recycling of hinge-primer binding sites, the reaction finally results in DNA product accumulation for specific detection. The “X” in the primer represents a blocking modification for the Bst DNA polymerase. Examples are a dSpacer (abasic furan) or an C12-Spacer (hexaethylenglycol). Optionally, an additional outer primer can be applied to increase the speed of initial single strand template generation.

    Article Snippet: LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template.

    Techniques: Generated, Binding Assay, Blocking Assay, Modification